来自美国加州神经科学中心和弗吉尼亚大学的研究人员表示，采用最新的高灵敏、高分辨“染色体错误定位技术”对iPS细胞系进行查错研究，认为iPS细胞很少有DNA结构性突变，逆转录因子受到了抑制。 + v$ `1 [* L$ X" l& e# b0 J+ R; J ! r. I5 f2 @# _5 q* [# nStem Cell Reprogramming Technique Is Found To Be Safer Than Once Thought% D4 b& p: I2 b
Thursday, October 06, 2011 - Stem Cell Research News + v8 C, S3 h% e' k# Z# s3 n h
, n. {& `2 n- m0 M& ]: d % _& W; K" B: X. @+ P Kristin Baldwin 3 ?. A3 |9 _. B9 f. L ) L" ]' M2 i h6 |9 M2 W q6 W2 |
, B3 q3 V& N: o8 A" Q- M- iStem cells made by reprogramming patients’ own cells might one day be used as therapies for a host of diseases, but scientists have feared that dangerous mutations within these cells might be caused by current reprogramming techniques. - A. x% s7 O/ u. c4 U% _
4 ~. y e! n. D% ?A sophisticated new analysis of stem cells’ DNA finds that such fears may be unwarranted. 3 E' x1 x9 s9 r" I3 g- ^: P # f+ v9 h% J- R0 O* \“We’ve shown that the standard reprogramming method can generate induced pluripotent stem cells that have very few DNA structural mutations, which are often linked to dangerous cell changes such as tumorigenesis,” said Kristin Baldwin, associate professor at the Scripps Research Institute’s Dorris Neuroscience Center (La Jolla, Calif.) and a senior author of the report. 3 u2 W( j( ^9 N" g7 J: Y0 }
* P$ I+ T5 l8 Y- D/ V0 T3 }For this study the Baldwin lab collaborated with a genomics and bioinformatics expert, Ira M. Hall, an assistant professor of biochemistry and molecular genetics at the University of Virginia who is co-senior author.6 R- T* P7 S! j/ {0 M5 S" ?( S
6 L& h% i6 c' B) M O6 QThe induced pluripotent stem cell (iPSC) technique was first described in 2006. It requires the insertion into an ordinary non-stem cell of four special genes, whose activities cause the cell to revert to a state like that of embryonic stem cell. In principle, iPSCs may be used to repair diseased or damaged tissues, and because they are made from a patient’s own cells, they shouldn’t provoke an immune reaction. % h- `; w7 f6 `2 l3 s* M" T
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But recent studies have found unacceptably high levels of mutations in iPSCs derived from adult human cells. That has led to widespread suspicion that the reprogramming process is largely to blame.2 u+ k$ ?1 l7 ^1 M
: V' r3 G0 K1 \. L. dIn the new study, the researchers set out to investigate this issue using the latest chromosomal error-mapping methods. ) u2 J. y# n: N2 v' i ) p5 {5 s( t0 A! t' Q“The techniques that our University of Virginia colleagues brought to this study are much more sensitive than anything else that’s available right now,” said Michael J. Boland, a research associate in the Scripps Research Baldwin lab and co-first author of the paper. - e5 c" Z% J g/ ?3 v6 r: }2 Z
; ^* G0 g; b7 \! q1 uThe new methods included a high-resolution version of a DNA-error-finding technique known as paired-end mapping, and an advanced algorithm, “HYDRA,” for handling the voluminous mapping data.# J8 P8 Q: R0 }
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To generate the iPSCs, the team followed the standard, four-gene reprogramming procedure, but sought to minimize other potential sources of DNA mutations that might have influenced some previously reported results. The donor cells they selected were not decades-old human skin cells, but relatively error-free fibroblast cells from fetal mice. The researchers also kept these fibroblast cells only briefly in lab dishes before reprogramming them.( T/ F8 z# U* [$ ]3 D
" w- K/ Z: \: `; \' |When the team members analyzed these iPSCs they used two strategies to distinguish which mutations were present in rare donor fibroblast cells and which were newly acquired during reprogramming. Their advanced techniques also allowed them to find more kinds of mutations, across a wider range of the genome, than ever before. Yet instead of finding more mutations, they found almost none. ; X! s k2 G) g% `* j" H( ]5 @) z
“We sequenced three iPSC lines at very high resolution, and were surprised to find that very few changes to the chromosomal sequence had appeared during reprogramming,” Boland said.% r8 C# s4 ]# w9 \6 F* `
$ |, t' n4 F) E* m: V8 j3 OEach of the iPSC lines contained only a single mutation that probably originated from the reprogramming process; two affected genes while the other appeared not to. Mutations inherited from the donor fibroblast cell were present in one pair of lines, while a second line “inherited” none. 3 m0 Z7 a4 e: m# m ' s1 x, K; ~3 \1 p$ p8 iThe researchers were particularly cheered by the complete absence of new “retroelement transpositions:” mutations caused by retrovirus-like sequences that burrowed into the mammalian genome long ago that can become active again in certain cell types. , p* ~% N& }% O
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All cells have ways to suppress these retroelements, but the suppression mechanisms in normal cells are different from those in stem cells, so the researchers had worried that retroelements would be allowed to escape suppression during the transition to a stem cell state. While no previous surveys of iPSCs could detect these mutations, this study showed that despite very sensitive detection of controls, no retroelements had become active during reprogramming. # O: y/ m, e0 k5 z' M, t, i# K% \* Z 2 O3 X9 j, O+ V“That was is very encouraging, because retroelement mutations can be very damaging to the genome,” Boland said./ y4 O3 G3 q( | C8 ]/ x
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Some of the mutations seen in human iPSCs in previous studies might have been due to incomplete reprogramming that impaired the cells’ DNA-maintenance mechanisms. In this study using mouse iPSCs, however, there was no doubt that a complete reprogramming to an embryonic state had occurred: all three iPSC lines were used to produce live, fertile mice, in work that Boland, Baldwin, and their colleagues described in Nature in 2009. 7 ~! f- u& u Z0 F! p9 J9 G7 O# i
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“The mice generated from these cells have survived to a normal lab-mouse lifespan without obvious diseases that might arise from new DNA mutations,” Baldwin said.4 F1 C( B2 c4 i3 O2 d' v$ R% g y
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Her lab now is trying to determine whether a reprogramming method similar to the one used with mouse iPSCs in this study could also yield relatively error-free human iPSCs. 3 K8 h% S% i& u% w9 @' E& Z/ I d
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“If our results with these mouse cells are applicable to human cells, then selecting better donor cells and using more sensitive genome-survey techniques should allow us to identify reprogramming methods that can produce human iPSCs that will be safer or more useful for therapies than current lines,” she said. s& n* T0 {5 G
1 }# f" L9 r; u/ W" J2 pCitation: “Genome Sequencing of Mouse Induced Pluripotent Stem Cells Reveals Retroelement Stability and Infrequent DNA Rearrangement during Reprogramming;” Kristin Baldwin, Ira M. Hall, Aaron R. Quinlan, Michael J. Boland, et al.; Cell Stem Cell, 7 October 2011.) h1 S! B: O3 [7 O6 M
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Contact: Kristin Baldwin, 858-784-9466, kbaldwin@scripps.edu6 X( r X6 `8 r0 ^' }
"Some of the mutations seen in human iPSCs in previous studies might have been due to incomplete reprogramming that impaired the cells’ DNA-maintenance mechanisms”; G& H, M; S# M- I N! C+ W4 s
文中说道hiPS由于重编程不完全，使得细胞“DNA维持机制”无法运行。楼主能不能帮忙解释一下这个机制啊？多谢了。看到这篇文章还是很高兴的，感觉最近iPS没有以前那么火爆了，不知是不是都忙着做其机制去了！如果真是文中所说的这样，我们对hiPS的安全性问题又可以重新思量一下了！作者: naturalkillerce 时间: 2011-10-8 23:41
回复 开心果王 的帖子. j* z8 O1 P; _6 z5 J6 C! E$ r
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首先文中说的是一些hiPSc突变可能是由重编程不充分或者说不完全，使得细胞DNA维持机制无法运行，即“是可能”，不要忽略这个词语。所谓细胞DNA维持机制无法运行就是指当DNA出现断裂或损伤后，各种DNA修复机制发生不能正常运转，导致细胞DNA复制受损，而这种情况是有可能在重编程过程中未严格遵守标准重编程程序导致一些DNA突变，而这些突变损害了DNA的修复机制。作者: naturalkillerce 时间: 2011-10-9 19:00
作者：Ira M. Hall 来源：《细胞—干细胞》 发布时间：2011-10-12 ; r/ b0 _, b0 P! N. V
/ O; r7 S' W( G h* H3 ?4 } iPSC基因组稳定性良好 - Z% K# Y. |* t; O8 v$ \! U+ Y
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美国研究人员通过对小鼠诱导多能干细胞的基因组测序，发现重编程过程不会增加DNA重排或逆转录因子转座的风险。该研究成果发表于10月4日的《细胞—干细胞》（Cell Stem Cell）杂志上。+ R+ L ?/ N& P* ?
, a; X! ]. T- D诱导多功能干细胞（iPSC）绕开了胚胎干细胞研究面临的伦理和法律等障碍，因此在医疗领域的应用前景非常广阔。然而，如果大部分iPSC含有有害的遗传突变，则它们的生物医学应用将会大大受损。近期有芯片研究表明人iPSC中的DNA拷贝数变异较胚胎干细胞要多，暗示其他类的基因组结构变异也会频繁发生。& a. x# `( k1 d$ r' ?5 H
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于是，弗吉尼亚大学和Scripps研究所的研究人员利用全基因组末端配对测序，试图解答iPSC的生产是否会导致突变增加和基因组稳定性降低的问题。与之前的研究结果相反，研究小组在所检测的三个小鼠iPS细胞系中发现了非常少的自发突变，也未发现逆转录转座子插入。 - C, ^) l4 i* p* D1 W3 ~$ h( ] P 5 z7 l( ?5 F6 K. |+ S
作者认为，这些结果表明基因组稳定性在重编程过程中能够保持，且目前的重编程方法有可能产生无基因干扰突变的iPSC。尽管还需要进一步研究来确定人的iPSC是否也保持着相同水平的基因组稳定性，但小鼠研究的结果让他们信心大增。 ( N6 @( e7 q* P4 I0 i9 b , D9 [) j* N2 V; N! W' N- {在本研究中，研究人员使用了Illumina的Genome Analyzer II对三个小鼠iPS细胞系进行了全基因组测序，而对照细胞是小鼠胚胎的胎儿细胞群。两个iPS细胞系是来自同一个体细胞，而第三个是来自另一个供体细胞。 ! e7 ~5 A$ ~4 D: a& P2 l, { 4 G7 s N. ^& Z9 t/ L) U研究人员产生了每个基因组的10-12倍覆盖度。他们利用了一种名为HYDRA的算法，来寻找重排和逆转录转座子插入。该算法在末端配对定位模式下检测结构变异的断裂点以及转座子插入。4 M9 Q4 C- T) m7 k1 c, v
5 D1 d) _) h B7 F$ S" g: Q即使未找到多个拷贝数变异或基因组重排，但研究人员认为他们可能会发现转座子激活。因为重编程基因组时，这是一个全基因组的表观遗传重编程，在扰乱表观遗传状态时，转座子可能激活。然而，未有证据表明iPSC中的逆转录因子转座。 - D$ l3 d: |, s! W ! K" k3 r' M: M. M& q( h; E# u
让人奇怪的是，之前在人iPSC研究中发现的一些遗传改变也未在小鼠iPSC中找到，研究小组认为，这可能与重编程的程度有关，或是与重编程所用的体细胞类型相关。（来源：生物通 薄荷）! l0 V8 {: w/ Y( l0 H
